Differential expression of gluconeogenesis-related transcripts in a freshwater zooplankton model organism suggests a role of the Cori cycle in hypoxia tolerance

Gluconeogenesis (GNG) is the process of regenerating glucose and NAD+ that allows for continued ATP synthesis by glycolysis during fasting or in hypoxia. Recent data from C. elegans and crustaceans challenged with hypoxia show differential and tissue-specific expression of GNG-specific genes. Here we report differential expression of several GNG-specific genes in the head and body of a model organism, Daphnia magna, a planktonic crustacean, in normoxic and acute hypoxic conditions. We predict that GNG-specific transcripts will be enriched in the body, where most of the fat tissue is located, rather than in the head, where the tissues critical for survival in hypoxia, the central nervous system and locomotory muscles, are located. We measured the relative expression of GNG-specific transcripts in each body part by qRT-PCR and normalized them by either the expression of a reference gene or the rate-limiting glycolysis enzyme pyruvate kinase (PK). Our data show that of the three GNG-specific transcripts tested, pyruvate carboxylase (PC) showed no differential expression in either the head or body. Phosphoenolpyruvate carboxykinase (PEPCK-C), on the other hand, is upregulated in hypoxia in both body parts. Fructose-1,6-bisphosphatase (FBP) is upregulated in the body relative to the head and upregulated in hypoxia relative to normoxia, with a stronger body effect in hypoxia when normalized by PK expression. These results support our hypothesis that Daphnia can survive hypoxic conditions by implementing the Cori cycle, where body tissues supply glucose and NAD+ to the brain and muscles, enabling them to continuously generate ATP by glycolysis.


Introduction
The role of the Cori cycle or gluconeogenesis (GNG) has been well characterized in humans and other mammals, where products of glycolysis are utilized in the liver to resupply muscles and other critical tissues with glucose and NAD+, thus allowing glycolysis to continuously generate ATP during bursts of muscular activity or while in hypoxic or fasting conditions [1][2][3][4][5][6]. In some other vertebrates adapted to either periodic fasting [7] or hypoxia [8], the Cori cycle emerges as a key element of adaptation to environmental extremes. The GNG pathway utilizes lactate, generated through the anaerobic catabolism of pyruvate, as a precursor for glucose synthesis. This multi-step process is assisted by the respective glycolytic enzymes catalyzing the reverse reactions, except for the exclusive involvement of phosphoenolpyruvate carboxykinase (cytosolic PEPCK-C and mitochondrial PEPCK-M), fructose 1,6-bisphosphatase (FBP), and glucose 6-phosphatase in GNG [6] (Fig 1). The upregulation of GNG-specific genes, primarily PEPCK paralogs, in hypoxia and the role of hypoxia-inducible factor (HIF-1) in this upregulation have been reliably demonstrated in mammalian models [9][10][11]. However, little data is available on GNG and the Cori cycle in aquatic invertebrates including plankton crustaceans. Aquatic organisms are more likely to experience periods of hypoxia than terrestrial ones, as oxygen solubility and diffusion rates in water are low, and its availability is highly dependent on temperature, the respiratory activity of aerobic heterotrophs, and the non-biological oxidation of organic matter. The ability to maintain energy metabolism anaerobically sets the limits to both hypoxia and heat tolerance in aquatic organisms [12] including Daphnia [13]. Therefore, it is important to examine GNG as a possible mechanism of hypoxic survival to understand the ability of organisms to cope with episodes of high temperatures and low oxygen availability in their natural habitats.
Studies on the decapod shrimp Litopenaeus vannamei indicate tissue-specific expression of GNG-related genes is consistent with the role of GNG in providing glucose to fuel muscles during hypoxia. The hepatopancreas in crustaceans, an organ that is functionally analogous to the vertebrate liver, shows higher gene expression of enzymes encoding for pyruvate carboxylase (PC) [14], PEPCK-C and PEPCK-M [15], and FBP [16,17] relative to muscle or gill tissues. Importantly, this tissue-specific gene expression is further enhanced by hypoxia [14,15,17]. Furthermore, glucose-6-phosphatase, one of the rate-limiting enzymes of glycolysis, shows a reverse response pattern to hypoxia, where it is upregulated in the gills but not in the hepatopancreas [18]. These results are consistent with the operation of the Cori cycle to allow hypoxia tolerance, with the hepatopancreas completing the GNG phase of the cycle and supplying tissues like muscles with glucose and NAD+. We expect this mechanism is likely conserved across invertebrates, as a similar differential expression was observed in the nematode Caenorhabditis elegans [19].
We have previously analyzed differential gene expression in response to mild chronic and severe acute hypoxia in a classic model organism for aquatic ecophysiology, Daphnia magna [20], revealing that while only a small subset of abundant transcripts showed differential expression responses under mild chronic hypoxic conditions, many, including the GNG-specific PEPCK-C, showed upregulation in acute severe hypoxia. Aralar1, a mitochondrial carrier protein that transports aspartate from the mitochondria to the cytosol [21], also showed upregulation in hypoxia. The function of Aralar1 is critical for GNG, as it allows bypassing pyruvate as the starting point of the pathway by converting aspartate to oxaloacetate, a PEPCK-C substrate that converts to phosphoenolpyruvate in the cytosol.
For the Cori cycle ( Fig 1A) to be effective, compartmentalization must exist between the tissues accomplishing each branch of the cycle: the glycolysis (e.g., muscles) and gluconeogenesis (e.g., liver). In Daphnia, the likely recipient tissues supplied with glucose to maintain glycolysis in hypoxia are the central neural system and the locomotory muscles, both located in the head of the animal (Fig 1B). It is somewhat more difficult to pinpoint the tissues responsible for the GNG branch. There is no direct homology to the vertebrate liver or decapod hepatopancreas in Daphnia anatomy. However, gene expression profiles and responses to knockout data in Drosophila [22,23] and lepidopterans [24][25][26] indicate that gluconeogenesis is likely to be accomplished by the fat body, allowing the designation of the fat body as a metabolic "equivalent of the mammalian liver" [22]. In Daphnia, the fat body is located along the gut in the thorax and abdomen regions (Fig 1C), with little to none present in the head. This provides an opportunity to test gene expression separately in the glucose recipient and glucose donor tissues by simply separating heads from bodies.
The goal of this study was to examine the differential expression of GNG-related enzymes PC, PEPCK-C, and FBP and the Aralar1 transporter separately in the head and body of hypoxia-challenged vs. normoxic control Daphnia. We hypothesize that transcripts of GNG ratelimiting enzymes should be more abundant in the body than in the head and be up-regulated in hypoxic conditions (Fig 1). To quantify the relative abundance of GNG-related transcripts, we used both a reference gene unrelated to glucose metabolism and a rate-limiting glycolysis enzyme, pyruvate kinase (PK), as normalization controls. The use of PK as the normalization control thus reveals the relative expression of the two opposing Cori cycle pathways in the heads and bodies of Daphnia.

Daphnia clones and culture
Daphnia magna stocks used in this study were obtained from the Basel University Daphnia Stock Collection (Basel, Switzerland) and maintained locally since 2016. The IDs of the four stocks used were FI-FSP1-16-2, GB-EL75-69, HU-K-6, and IL-M1-8; details of the provenance of these D. magna clones are supplied on Table 1 in Ekwudo et al. 2022. Hereafter, we refer to these clones by the first two letters of their Basel stock IDs. Previous longevity studies indicate that these clones differ significantly in their lifespan and acute hypoxia tolerance, with FI and IL being the short-lived, hypoxia-tolerant clones, and GB and HU showing higher longevity but lower hypoxia tolerance [20]. Stocks were maintained in modified ADaM zooplankton medium (Ref. [27]; https://evolution.unibas.ch/ebert/lab/adam.htm) at the density of one adult Daphnia per 20 mL, at 20˚C under 16h:8h L:D photoperiod and fed green alga Scenedesmus acutus Meyen (current nomenclature Tetradesmus obliquus (Turpin) M. J. Wynne) at the concentration of 10 5 cells per mL per day. Newborn individuals of each of the four clones were collected within 24 h of birth and placed in groups of 10 in 100 mL jars, with the density reduced to one Daphnia per 20 mL at maturity (day 6-8), and maintained with food added daily, water changed, and neonates removed twice weekly. The four clones used were characterized by their lactate:pyruvate ratio in normoxic and acute hypoxic conditions, as described below.

Acute hypoxia exposure and lactate: Pyruvate ratio measurements
Daphnia females maintained as described above until the age of 15-25 days were randomly assigned to control and hypoxia treatments. Hypoxia treatment individuals were placed individually in 10-mL screw-cap vials filled with 20˚C ADaM water deoxygenized to <1 mg O 2 /L by intense bubbling with nitrogen. Oxygen concentration was maintained by an Extech DO210 dissolved oxygen meter. Simultaneously, the control Daphnia were transferred into fresh vials containing fully oxygenized (>8 mg O 2 /L) ADaM. No food was added to either the hypoxia or control vials. Daphnia were harvested after 12 hours of exposure. Typically, no Whole Daphnia females from either control or acute hypoxia treatments were homogenized in 100 μL of RO water using a bead beater with 0.15 mm ZrO beads and centrifuged for 4 min at 4˚C. The supernatant was then used to determine lactate and pyruvate concentrations using CellBioLabs1 colorimetric kits (Cat. #s MET-5012 and MET-5125, respectively), according to the manufacturer's protocol, scaled down to 50 μL reactions, each containing 20 μL of supernatant. Absorbance was measured in 384-well plates on a BioTek Synergy plate reader at room temperature at 490 and 570 nm for lactate and pyruvate, respectively. In parallel, soluble protein content in the supernatant was measured by the Bradford method to normalize lactate and pyruvate concentrations per mg of protein.
Based on the results, we then selected the hypoxia-tolerant clone IL, characterized by the highest lactate:pyruvate ratio in hypoxia, for further qRT-PCR analyses.

Differential gene expression quantification
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) from the heads and bodies of 15 days old D. magna females (clone IL) exposed to acute hypoxia treatment or normoxia as described above. 10 females per sample were used for the RNA extraction, and four such samples were prepared for each condition. The RNA concentration was measured by Nanodrop, and samples were diluted to a final concentration of 20 ng/μL in RNase-free water. qRT-PCR was done using the qScript One-Step SYBR Green qRT-PCR Kit (Quantabio) according to the manufacturer's protocol, using gene-specific primers (Table 1). A total of 80 ng of RNA was used as input in a 20 μL reaction mixture, and all the reactions were carried out using an Illumina Eco Real-time PCR system with each treatment represented by four to five biological replicates, each in two technical replicates. The cDNA synthesis was performed at 50˚C for 10 minutes using the primers specific for the genes (Table 1), followed by polymerase activation at 95˚C for 10 minutes. PCR cycles were carried out at 95˚C for 10s, followed by 60˚C for 20s, for 40 cycles. Fluorescence signals (Channel 1; for SYBR Green) were detected after each cycle. The reference genes were chosen following a previous study [28]. We tested D. magna orthologs of the following proposed internal reference genes for constant expression: X-box binding protein 1 (Xbp1), TATA-box binding protein (Tbp), and syntaxin-16 (Stx16). Among these, Xbp1 showed no difference in expression between the control and hypoxia treatments and was thus chosen as the reference gene. The quantification cycle (Cq) values for each gene expression were determined from the amplification curves at a threshold value set at 0.02, and the relative expression of GNG-related genes was expressed as -ΔCq [29]. For further comparison relative to the pace of glycolysis, corresponding gene expressions were also normalized against that of the pyruvate kinase (PK), which catalyzes one of the rate-limiting steps of glycolysis in the muscles. Table 1 qRT-PCR primers used for GNG-related and reference genes. Xbp1, X-box binding protein 1; Tbp, TATA-box binding protein; Stx16, syntaxin-16; Arlar1; FBP, fructose 1,6-bisphosphotase; PEPCK-C, cytoplasmic phosphoenolpyruvate carboxykinase; PC, mitochondrial pyruvate carboxylase; PK, pyruvate kinase, muscle isoform.

Data analysis
Lactate and pyruvate content and qRT-PCR data were analyzed using Residual Maximum Likelihood (REML) ANOVA using JMP (Ver. 16, SAS Institute 2016), with protein-normalized lactate and pyruvate concentrations and their ratio, or relative expression, respectively, as the response variables. The main effects in the model were clones and acute hypoxia exposure. The date of measurement was included in the model as a random block effect. For qRT-PCR data, the biological replicate was included in the analysis as a random block effect.

Lactate and pyruvate content
Here we examined the differences in protein-normalized concentrations of lactate and pyruvate and their ratios under hypoxic and normoxic conditions among four D. magna clones (Table 2, Fig 2). Lactate concentration significantly increased in hypoxia but showed no evidence of interclonal differences, while pyruvate concentration did not increase in hypoxia but showed a strong clone effect, with the IL clone demonstrating the lowest levels of pyruvate. Consequently, the lactate:pyruvate ratio showed both clonal and hypoxia effects, with the increased lactate:pyruvate ratio after exposure to acute hypoxia being largely due to the increase in lactate concentration, whereas interclonal differences were largely due to differences in pyruvate concentration. There was no evidence of interaction effects between clones and hypoxia in either lactate or pyruvate concentrations or their ratio (Table 2).

Differential gene expression
The results of differential expression analysis by qPCR were somewhat different depending on which transcript was used for normalization: the carbohydrate metabolism-independent housekeeping reference gene Xbp1 or the glycolysis rate-limiting PK (Fig 1), even though PK itself showed no significant differences between either oxygen levels or body parts (Table 3, Fig  3). Normalization of GNG-specific transcripts by Xbp1 reflects general levels of expression; normalization by PK reflects the relative activity of the GNG vs. glycolysis branches of the Cori cycle. Of the four GNG-related transcripts tested, the transport protein Aralar1 transcript, contrary to predictions, showed a slight downregulation in hypoxia. Differences in PC expression between treatments were not significant regardless of which reference gene was used for normalization ( Table 3). The other two transcripts, PEPCK-C and FBP, showed hypoxia-related differential expression. Both PEPCK-C and FBP were upregulated in hypoxia when normalized by Xbp1 (tentatively significant after multiple test correction); FBP was both upregulated in hypoxia and in the bodies, relative to the heads, as predicted, with the hypoxia upregulation being stronger in the head than in the body (Table 3; Fig 3).

Discussion
The Cori cycle is an adaptation to intense bouts of muscular activity in larger animals, where it is difficult to supply sufficient oxygen for oxidative phosphorylation in muscles during peak ATP demand [5]. However, this is a costly way of generating ATP, as for every mole of ATP generated by glycolysis in the muscles, three moles are spent in the GNG branch of the Cori cycle, making it unsustainable in the long term. However, even small organisms may have to resort to GNG to regenerate glucose in hypoxic conditions. For example, zooplankton organisms like Daphnia must continuously swim (typically upward) to avoid hypoxic layers or spots within a lake or pond, and this is likely a subject for selection. In Daphnia and other cladocerans, the head contains major locomotory muscles, namely the ones driving the 2 nd (swimming) antenna, in addition to the mitochondria-rich, ATP-consuming central neural system (Fig 1B and 1C; Ref. [31], Fig 1). Thus, the head tissues and organs are likely to be the primary consumers of glucose synthesized by GNG in conditions when glycolysis is the main source of ATP. While the "classic" mammalian GNG-active organs, the liver and kidneys, do not have direct counterparts in Daphnia anatomy, we hypothesize that the fat body, located in the thorax and the abdomen, may be the main site of GNG. Lactate and pyruvate metabolism, including the starting points of gluconeogenesis such as the conversion of pyruvate to oxaloacetate by PC, is known to be active in adipose tissue in both mammals and arthropods [32]. Therefore, we hypothesize that the "body" (thorax and abdomen) may be the donor of glucose produced by GNG in the Cori cycle operating in Daphnia Table 3

Normalized by Xbp1
Normalized by PK  under hypoxic conditions, suggesting differential expression of the GNG and glycolysis-related genes in the body and the head. And a further prediction made was that acute hypoxia should augment this differentiation. These predictions were tested in a qRT-PCR experiment, measuring GNG-related transcript abundance separately in the heads and bodies of individuals from the IL Daphnia clone. We selected this clone from a panel of four as it was the one characterized by the lowest pyruvate accumulation in tissues, hypothetically indicating the utilization of pyruvate for lactic fermentation and/or GNG. This clone has previously been demonstrated to be the most hypoxiatolerant of the four clones tested [20]. We observed a significant upregulation of PEPCK-C and FBP (but not of PC) in hypoxia in both body parts when normalized by a reference gene unrelated to carbohydrate metabolism (Fig 3). We also observed a significant upregulation of FBP, a rate-limiting GNG enzyme, in hypoxia and in the body relative to the head, when normalized by PK, one of the rate-limiting enzymes of glycolysis. We therefore conclude that body vs. head compartmentalization of the glycolysis and GNG pathways is likely necessary for survival in hypoxia. Furthermore, we predicted the hypoxia-by-body part interaction, with the hypoxia effect being stronger in the body than in the head (Fig 3); however, this predicted interaction effect was only marginally significant and did not survive multiple test correction.
It is yet to be tested if the same observation would be made with a less hypoxia-tolerant clone of D. magna (or in less hypoxia-tolerant species of zooplankton). Further studies would test if that variation in hypoxia tolerance, within and among zooplankton species, is maintained by the trade-offs between the ability to operate the Cori cycle in hypoxia and the GNGassociated costs of doing so when oxygen is abundant.

Conclusions
The regulatory responses to hypoxia are still being described in aquatic invertebrates used in the aquaculture industry and as models. In a hypoxia-tolerant clone of Daphnia challenged with severe oxygen deprivation, we observe upregulation of the rate-limiting gluconeogenesis enzymes, PEPCK-C and FBP. When examining the general tissue-specific localization of these genes, the upregulation of FBP is more pronounced in the body than in the head of Daphnia, indicating the potential role of the Cori cycle in sustaining glycolysis in the central nervous system and locomotory muscles. As these genes are found to be upregulated across hypoxia-tolerant aquatic invertebrates, it is our belief that hypoxia-induced Cori cycle activation, or a similar pathway, is conserved in invertebrates that are frequently challenged with hypoxia. We hope future studies will describe this effect more broadly, with the aim of identifying it as an additional toolbox for hypoxia survival.